Conformational dynamics of CYP3A4 demonstrate the important role of Arg212 coupled with the opening of ingress, egress and solvent channels to dehydrogenation of 4-hydroxy-tamoxifen

Abstract

Background Structure-based methods for P450 substrates are commonly used during drug development to identify sites of metabolism. However, docking studies using available x-ray structures for the major drug-metabolizing P450, CYP3A4, do not always identify binding modes supportive of the production of high-energy toxic metabolites. Minor pathways such as P450-catalyzed dehydrogenation have been experimentally shown to produce reactive products capable of forming biomolecular adducts which can lead to increased risk toxicities. 4-hydroxy-tamoxifen (4OHT) is metabolized by CYP3A4 via competing hydroxylation and dehydrogenation reactions. Methods Ab initio gas-phase electronic structural characterization of 4OHT was used to develop a docking scoring scheme. Conformational sampling of CYP3A4 with molecular dynamics simulations along multiple trajectories were used to generate representative structures for docking studies using recently published heme parameters. A key predicted binding mode was tested experimentally using site-directed mutagenesis of CYP3A4 and liquid chromatography mass spectroscopy analysis. Results Docking with MD-refined CYP3A4 structures incorporating hexacoordinate heme parameters identifies a unique binding mode involving ARG212 and channel 4, unobserved in the starting PDB ID: 1TQN x-ray structure. The models supporting dehydrogenation are consistent with results from in vitro incubations. Conclusions and General significance Our models indicate that coupled structural contributions of the ingress, egress and solvent channels to the CYP3A4 active site geometries play key roles in the observed 4OHT binding modes. Thus adequate sampling of the conformational space of these drug-metabolizing promiscuous enzymes is important for substrates that may bind in malleable regions of the enzyme active-site.

Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions

“…In combination with other approaches, MD simulations were conducted on CYP3A4 in a number of studies, which (1) suggested that the F-F′-loop (residues 211–218) defines the promiscuity and broad substrate selectivity of CYP3A4 [83], and Ser119, Phe205, Arg212, Phe213 and Phe304 are key residues that help orient substrates in the active site [47, 83–86]; (2) identified preferred substrate access/egress and solvent channels [87–90]; (3) clarified the mechanism of cooperative binding of diazepam and ketoconazole [84, 91] and the role of CPR in activating water channels [92]; and (4) characterized the membrane-bound state of CYP3A4 [57, 93, 94]. …”

Understanding the mechanism of cytochrome P450 3A4: recent advances and remaining problems

“…59-68 The first MD simulations were conducted by Park et al 59 to clarify the roles of solvent dynamics and the active site loops in the ligand binding process and heme iron coordination. They showed that the malleability of the F-F’ loop (residues 211-218) defines the broad substrate specificity of CYP3A4, and that its complex with the inhibitor metyrapone is stabilized not only via direct coordination to the heme iron but also through a hydrogen bond between the metyrapone carbonyl oxygen and the Ser119 hydroxyl.…”

The Molecular Basis for the Pharmacokinetics and Pharmacodynamics of Curcumin and Its Metabolites in Relation to Cancer

“…The enzymatic source responsible for the reduction of curcumin is, however, strictly cytosolic, whereas the reduction of hexahydrocurcumin to octahydrocurcumin occurs in both the cytosol and microsomes (Ireson et al, 2002). In Caco-2 cell transport experiments, Wahlang et al (2011) showed that itraconazole-mediated inhibition of cytochrome P450 CYP3A4, an isozyme that is replete Cancer Pharmacology of Curcumin in the liver and intestines (Yokose et al, 1999) and capable of detoxifying certain drugs by reduction (Shahrokh et al, 2012), results in an increased curcumin permeability coefficient (Wahlang et al, 2011). This finding is suggestive of alterations in reductive biotransformation of curcumin by CYP3A4, which manifests itself in differential curcumin transport kinetics as a result of a shifted curcumin:reduced curcumin equilibrium and corollary effects on transporter activity.…”